1. Field of the Invention
This invention relates to a method and apparatus for automating the detection of target nucleic acid sequences in biological-containing samples involving a sequence of physical and chemical reactions, and more particularly to a system for the exposure of, amplification of, and labelled-probe coupling to, a specific, known nucleic acid sequence. The invention is especially suited to the automated detection of single, specific genetic sequences present at random in multiple samples containing biological material without labor-intensive DNA extraction and purification procedures being performed separately on each sample.
2. Description of the Related Art
Instruments for synthesizing polynucleotides have made genetic probes available on a commercial basis. There are now four or more companies selling DNA synthesizers. Improved and more widely known DNA sequencing strategies have enabled researchers to contribute sequence information to the literature and gene databases. The sequence knowledge and the ability to synthesize polynucleotides of a specific sequence have led to the development of genetic probe diagnostics. Wherever a unique, organism-specific polynucleotide sequence is identified, it is possible to use a labeled, synthetic molecule of the unique sequence to determine the presence of the organism by hybridization of the unknown sample to the labeled sequence. This detection method involves hybridization between DNA:RNA hybrids or DNA:DNA duplexes. The probe is a single-stranded nucleic acid molecule complementary to a unique nucleic acid sequence of the gene being tracked. The probe is labeled with an identifying molecule and introduced to the test sample. Hybridization has been an important research tool, but its use is limited to a few clinical laboratories because of the time, skill and knowledge required of the technician performing the test. DNA probes are being used as commercial diagnostics for a few infectious or genetic diseases, but their individual cost is prohibitive for mass screening.
While the common laboratory procedure for hybridization binds the target DNA to a solid support, an alternative approach is solution hybridization or hybridization which requires individual column separation of the unbound, labeled probe for each sample. This invention uses a gel matrix as a solid support. It is not necessary to transfer DNA to a membrane filter after purification and amplification. This approach eliminates any loss of DNA that occurs during transfer. (Purrello and Balazs, 1983, Anal. Biochem. 128:393-397). This paper and all other papers and patents cited herein are hereby incorporated in full by reference.
Presently DNA preparation and amplification require labor-intensive protocols just as hybridization methods do. The only apparatus which automates DNA preparation is the Applied Biosystems Nucleic Acid Extractor, which will process sixteen tissue samples simultaneously in four hours. The sample must comprise homogenous tissue and already contain enough copies of the target DNA to be detected, i.e. about a million copies. The laboratory technician must then either fractionate the extracted DNA by gel electrophoresis or transfer the DNA to a solid support for detection by hybridization to a labeled probe. There is no laboratory apparatus or equipment currently on the market that automates hybridization so that it may be left unattended.
Suspending cells in agarose beads or cubes is a common laboratory procedure for preparing unsheared nucleic acid molecules for subsequent enzymatic modifications. (P. R. Cook, 1984, EMBO 3:1837-1842 and L. Van der Ploeg et al. 1984. Cell: 37:77-84). After solidification the agarose beads or cubes are subjected to extensive treatment with detergent, protease and salt. It is possible to remove all cellular constituents except DNA because the pores in the agarose matrix are large enough to allow rapid diffusion of proteins and other small macromolecules while quantitatively retaining genomic DNA (Smith and Cantor, 1986, Cold Spring Harbor Symposium on Quantitative Biology 51:115-122).
FMC Bioproducts, Rockland, ME, has a nonradioactive-label for DNA in which their product information states that the labeling is done directly in diluted, remelted agarose. This protocol allows electrophoretic fractionation of DNA in agarose and then quick and easy preparation of specific probes (Resolutions 1987 Newsletter 3(2):1-2). FMC also markets a new grade of agarose certified for reliable restriction endonuclease activity. Many other examples exist where research scientists are performing enzymatic modifications on DNA in agarose. D. Persons and O. Finn, (Biotechniques, 1986, 4:398-403) reported primer extension of cDNA on a poly A+ RNA template using a reverse transcriptase in remelted agarose. The method and device of this invention also involves primer extension with polymerase enzymes in agarose.
The above applications using agarose demonstrate its unique properties for DNA manipulations. Agarose is the least charged subcomponent of agar, a mixture of polysaccharides from red algae. It is usually the gel of choice for electrophoresis of particles larger than 5-10 nm in radius. Since agarose is an alternating copolymer with variable amounts of several groups modifying the basic repeating structure, one theory suggests that double helices form during agarose gelation and the double helices cross-link and aggregate to form suprafibres. (P. Serwer, 1983, Electrophoresis 4:375-382). An agarose gel is more rigid than a polyacrylamide gel of the same concentration and an agarose gel has pores larger than the pores of all but the most cross-linked polyacrylamide gel.
Another difficulty that some scientists are trying to address is how to extract DNA from various mixtures in which organisms exist and in which they need to be detected. Human tissue, food products, environmental sources of water, sludge, or soil are among the diverse mixtures in which monitoring of genetic, identifying codes is desirable. If the samples, regardless of origin, could be treated by a standard process in a common apparatus without interference with the nucleic acid identification, then the need for prior nucleic acid extraction and purification steps for identification by hybridization protocols in current laboratory use would be eliminated.
Current practice for nucleic acid identification is done by dot blotting, colony hybridization, Southern transfer or "in situ" either in agarose or in tissue sections with microscopic identification of signal. Most of the hybridizations are performed for research purposes, and some are performed for diagnostic purposes. To recover a genetically altered cell or microbial strain it has been necessary to grow the cell or strain selectively. Direct gene-tracking eliminates the growth component requirement needed when enumerating counts. The processing of DNA or RNA in a solid matrix allows sample preparation, and target DNA amplification and identification of specific sequences without manual transfer of the sample during sequential treatments. The automation of sample-processing will bring accelerated growth to gene-monitoring of microbial, environmental, plant and animal samples.
Immunodiagnostics are commonly used to identify organisms directly by antigenic determinants or to identify individuals by their antibodies which are produced by exposure to the antigen. The same problem is encountered with antigen identification as with DNA probes, i.e. the organism must be cultured if it is not present in sufficient numbers for detection. There is no in vitro method to amplify antibody-binding antigens accurately like there is with primer extension gene amplification. Low population targets in a mixed background cannot be identified immunologically. The gene amplification in vitro has given DNA probes the potential to outperform immunological detection. The accuracy, sensitivity and quantitation potential of DNA probes will make them the diagnostic of choice.
An automated system for simultaneously detecting target nucleic acid sequences from multiple samples must accommodate several different steps and varying reaction conditions. It must be constructed to change reagents and solvents automatically for each stage and monitor time, temperature and pH. If tests are automated and the same apparatus that performs one test for multiple samples in one run could be used for many different tests by changing a few selected reagents, the cost of gene detection would be relatively inexpensive and the system would supersede other methods because of its speed and preciseness.
In order to have enough gene copies for detection, present methods rely on selective cultivation of the organism which takes days to weeks depending upon the organism. A selective DNA amplification technique has been practiced whereby synthetic primers are annealed to single stranded or denatured, double-stranded nucleic acid target sequences and polymerase molecules incorporate nucleotides that replicate a portion of the nucleic acid extending from the primers. Using excess primers in pairs bordering a target sequence in a way that each polymerase extension includes sequences that are complementary to the other primer sequence is a method now termed polymerase chain reaction (PCR) (see U.S. Pat. Nos. 4,683,195 and 4,683,202). This method continues in repetitive rounds of replication until the target sequence has been amplified by a factor of more that 10 million. Saiki et al. reported that a thermostable DNA polymerase improves the specificity, yield, sensitivity and length of products that can be amplified (Saiki, R. K., D. H. Gelfand, S. Stoffel, S. J. Scharf, R. Higuchi, G. T. Horn, K. B. Mullins, and H. A. Ehrlich, Science, 1988, 239:487-491).
A selective gene amplification protocol that can duplicate a single copy of a nucleic acid target in vitro to a sufficient number of copies that can be detected over non-specific background binding with a labeled hybridization probe is the level of sensitivity that will enable easy screening of multiple samples. The accuracy of a gene detection is assured by labeling a probe complementary to a polynucleotide sequence between the two primer sequences for the purpose of hybridization identification. Thus, even if the primers had amplified non-target sequences because of duplicity of sequence or mismatch, the label would only be detected that bound to the target sequence.
The ability to amplify a single target DNA and/or RNA sequence enough to detect it without the cultivation of cells or organisms will simplify gene detection and attempts to automate it. Saiki et al. reported that PCR detects a single copy of target DNA present in one in 1.5 million cells. There is no reason to doubt that gene amplification by primer extension will detect a target DNA segment present at one copy per organism in the starting material. The ability to then quantify how many original copies or organisms there were per sample before amplification will make mass sampling and fate-monitoring possible by hybridization detection. Quantifying methods depend upon diluting the amplified gene so that individual signals are enumerated or intensity of total signal matches that of a known standard concentration.
Using the aforementioned gene amplification protocol, the presence of HIV-1 in peripheral blood mononuclear cells (PBMC) was determined by in situ hybridization to DNA from the PBMC's without prior cultivation of them (Ou, C., S. Kwok, S. Mitchell, D. Mack, J. Sninsky, J. Krebs, P. Feorino, D. Warfield, and G. Schochetman, Science, 1988, 239:295-297). This direct detection method reduces the time to three days from the three to four weeks required for cell cultivation and virus isolation. The polymerase chain extension technique started with DNA isolated from PBMC's, repetitively amplified the target DNA in solution, and analyzed bands on an autoradiogram produced by gel electrophoresis of restriction enzyme digests of the target DNA bound to end-labeled radioactive probe molecules.
Accordingly, the invention aims to provide a system for automated gene identification of multiple samples, which prepares nucleic acids in the samples for testing, sufficiently amplifies target nucleic acid sequences and accurately detects their presence or absence in the samples.
Another object of the invention is to provide such a system which is adaptable to dispensing different quantities of different reagents.
Yet another object of the invention is to provide such a system wherein the reagents and solutions are forced through jet spray manifolds to evenly spray the matrices stacked in the reactor and diffuse through the matrices aided by gravity flow.
A further object of the invention is to provide such a system wherein airflow and heating regulate temperature and humidity.
A further object of the invention is to provide a system which can accommodate partial capacity loads, i.e., fewer matrices per run, or that can accommodate more than one probe per run.
A further object of the invention is to provide an automatic process and apparatus allowing identification of nucleic acid sequences that have been embedded or fractionated in a matrix whether or not prior extraction or purification of DNA has been performed in the invention.
A still further objective of the invention is to provide such a system wherein the temperature, time, pH, humidity, and pressure are monitored throughout the detection process.
Other objects and advantages of the invention will be more fully apparent from the ensuing disclosure and appended claims.